SUMMARY

Most animals, including reptiles, lower body temperature
(Tb) under hypoxic conditions. Numerous physiological and
behavioural traits significant to the regulation of Tb are
altered by hypoxia in ways that suggest an orchestrated adjustment of
Tb at a new and lower regulated level. We examined this
matter in bearded dragons, Pogona vitticeps, a species of reptile
that naturally exhibits open mouth gaping at high temperatures, presumably in
order to promote evaporation and thus prevent or avoid further increases in
Tb. The threshold for the onset of gaping (assessed as the
temperature at which lizards spent 50% of their time gaping) was reduced from
36.9°C in normoxia to 35.5°C at 10% and 34.3°C at 6%
O2. The overall magnitude or degree of gaping, measured
qualitatively, was more pronounced at lower temperatures in hypoxia. Females
consistently had lower gaping threshold temperatures than did males, and this
difference was retained throughout exposure to hypoxia. In addition to gaping,
evaporative water loss from the cloaca may also play a significant role in
temperature regulation, since the ambient temperature at which cloacal
discharge occurred was also reduced significantly in hypoxia. The results
reported herein strongly support the view that hypoxia reduces temperature
set-point in lizards and that such changes are coordinated by specific
behavioural thermoeffectors that modulate evaporative water loss and thus
facilitate a high potential for controlling or modifying
Tb.

Introduction

The bearded dragon, Pogona vitticeps (Ahl 1926), is an agamid
lizard that naturally inhabits the inland regions of Australia. In the wild,
it prefers temperatures of approximately 33°C
(Melville and Schulte, 2001)
and will adopt behavioural and physiological strategies to achieve this
preferred body temperature. Lizards achieve these kinds of preferred
temperatures in the wild through shuttling between warm and cold environments
(Brattstrom, 1971), modifying
heart rates during heat and cooling
(Seebacher and Franklin,
2001), altering peripheral blood flow
(Grigg and Seebacher, 1999),
changing cloacal evaporative water loss
(DeNardo et al., 2004) and
via ventilatory mechanisms, manifesting as gaping or panting
responses (Heatwole et al.,
1973). Numerous lizard species open the mouth widely when heated.
The temperature at which this gaping occurs in some lizard species is often
well above the preferred temperature, acting as a last-ditch attempt to
survive nearly lethal temperatures (Veron
and Heatwole, 1970; Webb et
al., 1972). Many lizards, however, begin panting at temperatures
at or very close to their preferred body temperature
(Heatwole et al., 1973),
suggesting that ventilatory heat loss through evaporation is an important
route for fine-tuning body temperature (Tb) regulation in
these species. Australian bearded dragons are lizards that employ the latter
strategy, spending much of their time basking and gaping, rather than
shuttling back and forth between warm and cool environments.

Numerous physical factors have been shown to alter the temperature at which
lizards gape or pant. Panting thresholds have been shown to be altered by
dehydration (Parmenter and Heatwole,
1975), circadian rhythms (Chong
et al., 1973) and the rate or source of heating
(Heatwole et al., 1973). The
direction of these changes reflects the constraints of other physiological
processes on thermoregulation; to conserve water, lizards do not gape until
higher temperatures, and, since daily metabolic demand decreases at night in
many reptiles (Rismiller and Heldmaier,
1987), a concomitant decline in the threshold for gaping occurs as
the thermoregulatory set-point (Tset) is reduced along
with the reduced metabolic requirements
(Rismiller and Heldmaier,
1982).

It is well established that low oxygen causes many animals, including
reptiles, to lower Tb
(Wood and Gonzales, 1996).
Changes in preferred temperatures are often taken as proof of a change in
temperature set-point in the brain, since preferred temperature selection is a
behavioural and thus, ultimately, a neurophysiological phenomenon. Numerous
lizards (e.g. genera Iguana, Dipsosaurus and Anolis) select
temperatures in hypoxia approximately 10°C lower than normoxic preferred
temperatures (Hicks and Wood,
1985; Petersen et al.,
2003). If the Tset is decreased in hypoxia,
then the thresholds for any autonomic or behavioural response that results in
a decline or an attempt to decrease Tb should be
decreased. Since many lizards exhibit panting or gaping responses as ambient
temperature rises (Crawford and Barber,
1974; Crawford and Gatz,
1974; Crawford et al.,
1977; Heatwole et al.,
1973; Pough and McFarland,
1976), this is taken to imply that reptiles exhibit some degree of
control of Tb, albeit manifesting as a behavioural
response.

With respect to behavioural thresholds for thermoregulation, Dupré
et al. (1986) showed that the
evaporative cooling threshold is diminished in hypoxic lizards; however, no
attempt to assess the magnitude or persistence of the gaping response was
made. Lizards that gape or pant may do so intermittently. It would be fruitful
to assess the entire gaping response across a wide range of temperatures in
order to determine whether the response is sustained, is proportional to
temperature and the severity of hypoxia and is dramatic enough to have an
effect on Tb regulation.

Studying the control of Tb in lizards under hypoxic
conditions allows for the exploration of the existence of thermal threshold
responses and thus makes inferences regarding the presence of thermoregulatory
set-points. The objectives of this study, however, were to examine whether the
gaping behaviour was proportionately related to ambient temperature and
whether the threshold temperature at which gaping occurred was proportionately
lowered in hypoxia. Understanding these two questions will shed light on the
mechanism of thermoregulation in reptiles and other vertebrates and will
provide evidence for whether hypoxia elicits a reduction in a hitherto
seldom-studied behavioural response in reptiles. For the purpose of this
study, we examined the gaping response (i.e. simple mouth opening) rather than
panting (i.e. an altered breathing pattern). It is plausible that bearded
dragons actually adopt panting, where the breathing frequency rises and tidal
volume declines; however, assessing tidal volume accurately is difficult to do
in lizards that open their mouths. Since simply opening the mouth may achieve
a similar result of eliciting evaporative water loss across the mucosa within
the mouth and throat, it is not necessarily required that panting accompanies
gaping behaviour.

Materials and methods

Animals

Fourteen (eight male and six female) inland bearded dragons, P.
vitticeps, were used in this study. All were captive-bred individuals
ranging in age from approximately 6 to 12 months, as well as one adult lizard
(>5 years of age). The lizards ranged in mass from approximately 50 g to
220 g, with the adult weighing 350 g. The animals were fed a daily diet
consisting of crickets and mealworms and were provided with chopped vegetables
ad libitum. Tb in the home cages (using a non-contact
infrared thermometer) and body masses were monitored once a week at 11.00 h.
Lizards had access to a 100 W light bulb, which could allow their
Tbs to rise as high as 39°C or as low as 28°C in
the housing environment. Animal experimentation was approved by the Brock
University Animal Care Committee (ACUC Protocol #031001).

Experimental set-up

Throughout experimentation, an individual lizard was housed in a
24×24×40 cm clear acrylic box, which was blacked out on three
sides to prevent distractions and to minimise reflections. Once inside the
box, the lizard sat elevated 3.5 cm from the bottom on a perforated, clear,
acrylic platform, which allowed gas to be pumped into the box from a tube
below the platform and helped to collect urine and faeces. The box was located
within an environmental chamber (Thermo Forma; Marietta, OH, USA) to enable
changing temperatures between 30 and 40°C. A non-radiant heat source was
chosen in order to simplify the exposure regime. Since lizards reacted to
human presence, a small surveillance camera was affixed to the environmental
chamber inside wall and aimed towards the acrylic box to allow for observation
of the undisturbed lizard's behaviour on a video monitor outside the chamber.
An infrared (IR) thermal imaging camera (Mikron 7515; Oakland, NJ, USA) was
positioned on top of the box, looking down onto the lizard, to obtain body
surface temperature data. The IR imager was hooked up to a computer outside
the chamber to obtain computerised images. In order to allow for varying
oxygen levels inside the box, the IR camera rested on a set of `bellows',
sealed to the box with weather stripping, making the box relatively air-tight.
A small tube was inserted beneath the bellows into the box to allow for gas
sampling during hypoxic conditions to verify oxygen levels of 21, 10 and
6%.

Data collection

The animals were placed in the chamber to start experimentation in the
morning, preferably before feeding. Since it has been shown that lizards have
different gaping thresholds between night and day
(Chong et al., 1973), we
elected to perform the gaping measurements during the same time of the day,
between 10.00 h and 16.00 h. Lizards were given time to acclimate to the new
temperature, as well as for the box to reach hypoxic levels if necessary. This
usually required 20–30 min.

Assessing gaping behaviour

The lizard was observed for 15 min at each temperature of interest (30, 32,
34, 36, 38, 40°C), and the time spent gaping was recorded together with
the degree or type of opening of the lizard's mouth
(Fig. 1). Type I represented a
barely open mouth, Type II was a typical gape and Type III was a wide open
mouth, usually accompanied by a head-back posture, with the tongue partially
protruding and a puffing out of the throat. The observation periods were
started once the lizard's dorsal surface temperature was within 0.5°C of
the ambient temperature of interest. The only exceptions were the observation
periods at 40°C (in normoxia and 10% oxygen) and 38°C (at 6% oxygen),
when the animal was observed before it had reached ambient temperature. This
was due to the long time required for the animal to reach the highest
temperatures. The animals were observed sooner in order to prevent heat
damage, since at this temperature all animals were already gaping
maximally.

The three types of gaping categorised in this study. Type I was ascribed to
situations when the mouth was visibly, but barely, open (i.e. there was no
obvious sealing of the upper jaw with the lower jaw), which is readily
distinguished from a normal closed mouth. Type II was ascribed to situations
when the mouth was obviously open by more than a few millimetres. Type III was
easily distinguished from other types as the lizard's tongue was easily
visible and the throat was obviously distended due to the open mouth.

Assessing animal surface temperatures

The camera software, MikroSpec RT Version 2.1394 (Oakland, NJ, USA), was
set to capture an image of the thermal data every minute, up to a maximum of
500 frames. Image capturing was started upon the beginning of heating to
32°C (or 30°C at 6% oxygen). Images from the time during the 15 min
observation period were analyzed, and the lizard surface temperatures recorded
every minute. The mean temperature of a circular region of interest was
recorded from the lizard's back, head (between the eyes), tip of the nose,
tongue tip (if visible when gaping) and eye.

Experimental design and test conditions

The animals were each tested under normoxic and two hypoxic conditions (10
and 6% O2), which was accomplished by mixing air with nitrogen to
achieve the desired oxygen level. In all cases, gases entered the box at 5 l
min–1. Since no previous data were known on the effects of
hypoxia in P. vitticeps, initial observations were made of the
animals at oxygen levels between 5 and 10%. An oxygen level of 6% was chosen
as appropriate for this experiment, since this placed a significant enough
stress on the animal while not seriously risking damage to the animal after
the 6 h ofexposure required (i.e. lizards appeared distressed at levels below
6% O2 whereas at 6% O2 or above they remained calm
throughout the procedures). Throughout the trials, the oxygen level was kept
to within ±0.2% of the desired level. One animal was tested per day at
each oxygen level. Two to three weeks passed before the performance of another
experiment on the same individual at a new level of oxygen. At each level of
oxygen, lizards were tested at 4–5 different ambient temperatures
between 30 and 40°C (32, 34, 36, 38 and 40 for 21 and 10% O2
and 30, 32, 34, 36 and 38°C for 6% O2). Lizards were exposed to
these temperatures in a step-wise fashion, with the 2°C increments lasting
either 1 h each or as long as it took to achieve skin surface temperature
equilibration with ambient temperature. It usually took approximately 1 h for
the lizard's body surface temperature to come into equilibrium with the
environment. The highest temperature (40°C) was not used in the 6%
O2 group out of concern for the lizard's survival. Previous studies
have shown that lizards held at high temperatures under hypoxic conditions
will die (Hicks and Wood,
1985).

Data analysis

All values reported are means ±
s.e.m., unless otherwise stated. The
percentage time spent gaping at the different ambient temperatures was
initially analysed to determine a temperature at which 50% of the animal's
time was spent gaping. This was done by fitting individual Hill equations to
each animal using:
(1)
where Pgape refers to the percentage of time spent gaping,
Ta is the ambient temperature, ET50 is
the effective temperature at which 50% of the animal's time was spent gaping,
and the exponent, N, is Hill's constant (a larger N
indicates a steeper sigmoidal relationship between percentage gaping time and
Ta). The parameters that minimised the root mean square
error were determined using an iterative procedure facilitated by
Microsoft™ Excel's solver tool.

Data (either time spent gaping or surface temperatures) were analysed using
repeated-measures two-way ANOVA, with oxygen and ambient temperature as the
two treatments. On occasions where normality was not met, log transformations
were performed and the test repeated. In all cases, residuals from the
individual ANOVAs were examined to verify a normal distribution. If residuals
were non-normal, a non-parametric repeated-measures ANOVA of ranks was
performed. We were able to examine the effects of gender once we had
calculated the ET50s and N. On that occasion, we
used two-way repeated-measures ANOVA to test for significant
ET50 and N, with oxygen level and sex as
treatments. In cases where ANOVAs yielded significant effects,
post-hoc multiple comparisons were performed using the Holm-Sidak
method. All statistics were considered significant at P<0.05.

Results

General observations in hypoxia

Lizards initially responded to the test chambers by turning around and
searching their environments, settling down within 30 min, moving only
occasionally thereafter. At the lower levels of oxygen, lizards would often
close their eyes, appearing to fall asleep, though we did not make attempts to
quantify this. Most lizards defecated or urinated in the test chamber during
the course of observations (Fig.
2), even though they had been fasted for 16 h prior to
experimentation. This discharge led to a dramatic and often prolonged decline
in tail surface temperature (Fig.
2). Lizards (10/14=71.4% of the individuals) kept under normoxic
conditions first discharged faeces and urine (cloacal discharge) at
36.1±0.6°C. At 10% O2, the cloacal discharge temperature
(TCD; back surface temperature at which cloacal discharge
first occurs) fell significantly (P=0.02) to 34.4±0.8°C in
71.4% of lizards, and at 6% O2 the TCD was also
significantly lower (P=0.02) than normoxia at 33.2±0.4°C
(Table 1) in 78.6% of
individuals. There was no significant difference in the
TCD between 10 and 6% O2. The lizards that did
not exhibit cloacal discharge (21.4–28.6% of individuals) were not
factored into the calculations for TCD. Of those lizards
that exhibited cloacal discharge during exposure to temperatures between 30
and 40°C, 1.8±0.2 discharges occurred at 21% O2,
1.5±0.3 discharges occurred at 10% O2, and 3.4±0.4
discharges occurred at 6% O2. The latter value was significantly
higher than the values at 21% O2 (P<0.05;
Table 1).

Infrared thermal images of lizards at different ambient temperatures
showing Type II gaping (A), Type III gaping with the inside of the mouth
clearly visible (B) and cloacal discharge in two different lizards (C, in
hypoxia; D, in normoxia). In all cases, regional temperature differences can
be observed across the body surfaces. Note the different temperature keys to
the right of each image.

Gaping times in normoxia and hypoxia

Overall gaping time was significantly affected by inspired oxygen level
(P<0.001) and by temperature (P<0.001;
Fig. 3). In all cases, the
total gaping time increased sigmoidally at the higher ambient temperatures;
however, there was no significant interaction between oxygen and temperature
(P=0.112). The mean ET50 values for 21%, 10% and
6% O2 were 36.9±0.2°C, 35.5±0.4°C, and
34.3±0.4°C, respectively (Table
1), and the effect of hypoxia (10 and 6% O2) was found
to be significantly lower than normoxia. There was also a significant trend
for the Hill constant, N, to be higher in hypoxia, although this
effect was only significant at 6% O2 (P=0.05). N
was 69.8±11.0 at 21% O2, 78.4±10.8 at 10%
O2 and 113.4±15.6 at 6% O2
(Table 1). The appropriateness
of ET50 as an overall estimate of the threshold
temperature is demonstrated by the good fit (r2=0.873,
P<0.001) of the regression of ET50versus the total time spent gaping between 30 and 38°C (i.e. the
experimental duration).

Mean times (± s.e.m.) spent gaping
in lizards at three different levels of oxygen – 21% (filled circles),
10% (open circles) and 6% O2 (filled triangles) – during 15
min periods of observations of lizards that were in thermal equilibrium with
different environmental temperatures (30–40°C). The broken lines
represent Hill equations (see equation 1) fitted through the mean data. The
vertical dotted lines represent the mean time at which lizards spent 50% of
their time gaping (ET50) at each level of oxygen inspired,
as calculated from each lizard. The inset graph refers to the
ET50 values for male (filled circles) and female lizards
(open circles) at 21, 10 and 6% O2.

Effect of sex on thermal preferences and
ET50

Using a two-way repeated-measures ANOVA to test ET50
with oxygen and sex as the treatments, there was no significant interaction
(P=0.49), although sex and oxygen level each had a significant effect
(P=0.046 and P<0.001, respectively). Male lizards
exhibited significantly higher ET50 values than females at
all levels of oxygen tested (Table
1). Interestingly, this sex difference was also borne out in the
mean preferred temperatures of lizards in their home cages during the 3-month
period of experimentation; just as in the ET50 estimates,
there was a significant effect of sex on home cage preferred temperature
(P=0.02). Males exhibited a slightly, though significantly, higher
Tb of 35.2±0.17 versus
34.2±0.34°C in females.

Gaping type in normoxia and hypoxia

There was a significant effect of temperature on the percentage time spent
in Type I gaping (P=0.03), but no significant effect of oxygen
(P=0.31) nor a significant interaction effect (P=0.18). At
all three levels of oxygen tested, the percentage time spent in Type I gaping
gradually increased with increasing temperature before falling back down to 0%
at the highest temperatures (Fig.
4).

Mean times (± s.e.m.) spent engaged
in (A) Type I, (B) Type II and (C) Type III gaping at 21, 10 and 6%
O2. Data for 40°C are shown for comparison, although not
included in statistical analysis, since data points were not available at 6%
O2. † refers to a significant difference between
10 and 21% O2, and * refers to a significant difference between 6
and 21% O2 with post-hoc tests.

Type II gaping was significantly affected by inspired oxygen
(P=0.02) and ambient temperature (P<0.001). Furthermore,
there was a significant interaction between oxygen and temperature
(P<0.001). Type II gaping predominated at lower temperatures at 6%
O2. At all three levels of oxygen, Type II gaping initially
increased with higher temperatures before decreasing at the highest
temperatures (Fig. 4).

There was a significant effect of oxygen and temperature on the percentage
time spent in Type III gaping (P<0.001 for oxygen and
P<0.001 for temperature) and a significant interaction between
oxygen and temperature (P<0.001). Qualitatively, Type III gaping
was initiated earlier (i.e. at lower temperatures) in hypoxia than in normoxia
(Fig. 4). Indeed, at
temperatures above 36°C, significantly more time (>50%) was spent
engaged in Type III gaping at 6% O2 than at 21% O2.

Effect of body mass on gaping times

There was no significant effect of body mass on the
ET50 estimates at 21, 10 or 6% O2, despite the
large range of body masses examined (P=0.18, 0.29 and 0.45 and
r2=0.14, 0.09 and 0.05, respectively, determined through
linear regressions). Furthermore, body mass had no significant effect on
N estimates for 21 and 10% O2 (P=0.26 and 0.76
and r2=0.10 and 0.008, respectively); however, N
estimates were significantly and negatively correlated with body mass in the
lizards at 6% O2 (P=0.0005 and
r2=0.66), demonstrating that small lizards exhibited a
more rapid transition to continuous gaping as temperature increased.

Mean surface temperatures (±
s.e.m.) exposed to (A) 21, (B) 10 and (C) 6%
O2 during changes in ambient temperature ranging from 30 to
40°C. Shown are surface temperatures of the head, body, nose, eye and
tongue (when visible). The dotted lines represent the line of equality for
surface temperature and ambient temperature.

Effect of ambient temperature and oxygen on surface temperatures

Ambient temperature significantly affected all surface temperatures (body,
head, nose, eye; P<0.001 for all values), except for tongue
surface temperature, where there was a nearly significant difference
(P=0.062). Oxygen level had no significant effect on any of the
surface temperatures (except tongue) nor was there a significant interaction
between oxygen and temperature for either body, head, nose or eye temperature
(P=0.466, 0.10, 0.011 and 0.261, respectively;
Fig. 5).

The only meaningful significant effects were on the tongue temperature. To
simplify comparisons and account for some inter-individual responses, we also
examined the body surface–tongue surface temperature difference. Oxygen
level and ambient temperature had significant effects (P=0.011 and
P<0.001, respectively) on the body surface–tongue surface
temperatures, although there was not a significant interaction between oxygen
and ambient temperature (P=0.92;
Fig. 6). Overall, the
body–tongue difference was greater at 6% O2 than at 21%
O2, an effect that was most apparent at the lower ambient
temperatures.

Heating times in normoxia and hypoxia

Although not specifically controlled for, the times required to heat body
surface temperature from 30 to 38°C over the course of the entire
experiment were significantly affected by inspired oxygen (P=0.003).
It took 225.6±12.7 min in normoxia to warm body surface temperature to
38°C, 249.4±9.2 min at 10% O2, and 283.1±9.7 min
at 6% O2 (the latter value being significantly higher than the
normoxic value with post-hoc comparisons).

Body surface temperature minus tongue temperature at 21, 10 and 6%
O2 between ambient temperatures of 32–38°C. There was a
significant effect of oxygen and ambient temperature on this difference
(two-way repeated-measures ANOVA), suggesting that hypoxic conditions led to a
higher value at lower ambient temperatures. The dotted line represents an
extrapolation beyond 38°C in the 6% O2 group.

Discussion

The results of this study support the notion that the
Tb set-point for gaping is reduced in proportion to the
severity of hypoxia and that evaporative cooling in the bearded dragon seems
critical to normal Tb regulation. That an apparently
simple behavioural response such as gaping can be so elegantly controlled, in
both magnitude and duration, speaks volumes for the importance of
thermoregulation in this species. In terms of behavioural strategies for
thermoregulation, an argument could be made for the importance of this
response since the costs of gaping are relatively inexpensive, provided a
lizard is hydrated. In general, animal behaviour is believed to exhibit
optimality, such that the costs and benefits of a given behaviour balance out.
Provided adequate moisture is available, gaping or panting can be an effective
strategy that does not require the movement into or out of certain thermal
environments. Increased movement in the wild may lead to increased visibility
and thus increased risk of predation, so if subtle behavioural adjustments can
modulate Tb adequately, then the less expensive
behavioural strategies should prevail. Whether the altered gaping response to
hypoxia would occur in those lizard species that only utilise gaping and
panting as a last resort remains to be seen.

Various threshold parameters in bearded dragons as they pertain to the
appropriateness of thermoregulation. (A) The plot of ET50versus cumulative time spent gaping during experimental procedures
demonstrates that ET50 estimates provided a good fit
across all three levels of O2. (B) Cloacal discharge threshold
(TCD) in normoxia versus mean cage temperature
(see Materials and methods) showed no significant relationships. (C) A
significant relationship between ET50 in normoxia and mean
cage temperature was not apparent, although a significant intercept did occur,
suggesting that the ET50 tends to occur at temperatures
higher than preferred cage temperatures. (D) There was a significant
relationship between ET10 (the temperature at which the
lizards were gaping for 10% of the time) and mean cage temperature, although
no significant intercept, suggesting an isometric relationship between the two
variables.

Applicability of gaping thresholds to reptilian thermoregulation

It is possible that the wide range in body masses (50–350 g) used in
this study led to some variability in the data. We found, however, no
significant trends with body mass on the ET50 estimates,
which is consistent with Heatwole et al.
(1973), who found no effect of
size on the panting threshold in a related species, Amphibolurus
muricatus. Another possible concern is that we were only measuring
surface temperatures of the lizards rather than core Tb.
Since core Tb (which should be the regulated variable)
will always lag behind peripheral temperatures during changes in ambient
temperature, the percentage time estimates for gaping might actually be
underestimates since core temperature could still have been rising during our
assessments of gaping behaviour. Thus, the ET50 values may
be slight overestimates (since ET50s are negatively
correlated with total time spent gaping; see
Fig. 7), although this effect
should be consistent across all oxygen levels. The use of ambient temperature
as the reference point rather than a particular surface temperature may have
circumvented this problem. Furthermore, it could be argued that lizards
utilise peripheral thermal information in a feed-forward fashion to predict
changes in core Tb. If that were the case, the use of
peripheral temperatures in our experiments might be more relevant in the
assessment of gaping threshold.

During heating, the periphery would be the first to warm up and,
considering the fact that ET50s are always higher than
preferred Tb values in normoxia
(Fig. 7C,D), this suggests that
the majority of gaping occurs at or above the normal Tset,
acting as an important contributor to Tb regulation when
ambient temperatures exceed Tset. The fact that, in
normoxia, a regression of normoxic ET10versus
mean Tb in home cage yielded a significant regression
suggests that the gaping thresholds determined here are meaningful values with
respect to the individual lizard's normal preferred temperatures and that
gaping is not simply a randomly displayed behavioural response.

One final concern was that at the highest temperatures of
38–40°C, lizard surfaces did not come fully into equilibrium with
ambient temperature within the experimental time period. This suggests the
efficacy with which bearded dragons can defend a lower Tb
through increased evaporative cooling, since at 40°C lizards spent 100% of
their time gaping. Thus, although the surface temperatures (and thus also
Tb) were not in true equilibrium with ambient temperature,
this would not have affected the ET50 estimates, since the
maximum gaping efforts were already reached (i.e. values greater than 100%
would not have been possible and thus the Hill equations would not have been
affected).

Hypoxia reduces ET50 through a proportional regulator

The main hypothesis tested in this study, that hypoxia would lower the
gaping threshold in a proportional fashion, was supported by the data. The
central hypothalamic thermostat is thought to operate as a proportional
controller (Mrosovsky, 1990).
In other words, the further the regulated variable deviates from a set-point,
the larger will be the corrective response. In this case, as lizards are
warmed up, they gape for progressively longer periods of time. This regulator
responds, as well, to magnitude; lizards in hypoxia had a greater tendency to
exhibit the more pronounced Type II and Type III gaping at lower ambient
temperatures than in normoxia (Fig.
4).

One interesting result from this study is that the three levels of
O2 yielded a proportionate reduction in the gaping threshold.
Previous work examining the preferred Tb of hypoxic
lizards seemed to indicate that a critical level of hypoxia lower than 10%
O2 was required before a proportionate drop in
Tb would occur (Hicks
and Wood, 1985). There are no data on the preferred temperatures
of P. vitticeps in hypoxia; however, it is possible that the gaping
threshold responds slightly differently to low oxygen than set-point driven
behaviour that requires activity or movement (i.e. preferred temperature).
Strict or `precise' thermoregulatory behaviour has been thought only to occur
in reptiles when inhabiting `low-cost' environments with low risk of predation
(Huey and Slatkin, 1976). If the costs for thermoregulating (e.g. locomotory
costs or risk of predation) are too high, then precise thermoregulation will
not occur. In this context, gaping or panting can be viewed as a low-cost
strategy for thermoregulation, inasmuch as its instantaneous costs are low.
Thus, it might be expected that the threshold for gaping could more easily be
sensitive and responsive to factors that alter set-point than a locomotory
means of regulating Tb could.

Regional differences in surface temperatures

During the present experiments, lizards were gradually warmed up from a
temperature of 30°C (their surface temperature in the early morning at the
beginning of experiments) to a final temperature of 38 or 40°C (depending
on O2 level; see Materials and methods). The overall heating times
between temperatures of 30–38°C were progressively longer in the
hypoxic trials. This was probably due to their propensity to gape and exhibit
cloacal discharge at lower thresholds, and hence the augmented evaporative
cooling allowed for greater attempts to defend a lower core
Tb at the higher ambient temperatures. Whether other
thermoeffectors operate in a similar fashion remains to be shown. Despite the
slightly different heating times, most surface temperatures were not affected
by hypoxia, except for the tongue surface temperatures, where the changes that
occurred strongly suggest that some internal temperatures (i.e. brain) may be
differentially controlled in hypoxia.

Previously, Pough and McFarland
(1976) showed that brain
temperatures of lizards housed at temperatures greater than 40°C exhibit a
substantial difference from the body, a response that was not observed in dead
lizards held at similar temperatures. In extreme cases, lizard brain
temperature can be up to 6°C lower than Tb
(Crawford and Barber, 1974;
Warburg, 1965). It is tempting
to ascribe a physiological role (i.e. preferential blood flow that favours
brain cooling at high ambient temperature) for this response, although one has
yet to be shown. Since we were only examining surface temperatures, we cannot
comment on brain temperature or its regulation. Previous work by Webb et al.
(1972) showed significant
body–head temperature differences in a wide variety of lizards. We did
not see very large differences in the present study, although this could be
because surface temperatures are not as informative as internal temperatures.
Interestingly, a dragon lizard in the Webb et al.
(1972) study exhibited panting
at high temperatures (usually greater than 40°C), compared with P.
vitticeps in the present study. This could have been related to the
rapidly induced thermal changes, which is quite opposite to the present study.
Our study was looking at steady-state changes, where lizards had time to
equilibrate with their environmental temperatures, and, as such, we notice
lizards gaping at much lower temperatures.

We used body–tongue temperature differences to estimate the degree of
evaporative cooling. The difference between body surface temperature and
tongue temperature increases at higher ambient temperatures, suggesting a
greater degree of evaporative cooling at higher temperatures. It is also
apparent that the continuous gaping that occurred in hypoxia, combined with a
presumably higher ventilatory rate (i.e. an hypoxic ventilatory response), led
to a higher degree of evaporative cooling in the hypoxic lizards across all
the test temperatures. This is not surprising given that preservation of brain
function would be more critically challenged under hypoxic conditions. Small
changes in brain temperatures have been shown to produce large changes in
brain damage in either hypothermic or febrile animals
(Herrmann et al., 2003;
Kataoka and Yanase, 1998;
Katz et al., 2004;
Trescher et al., 1997); higher
temperatures exacerbate excitotoxic damage, and lower temperatures lead to
less damage.

Qualitative aspects of the gaping response

It is possible that using a non-radiant means for changing
Tb is not the most appropriate for lizards that tend to
bask in the sun. Indeed, Heatwole (1973) showed that thresholds for gaping
were less variable in radiantly heated individuals than in non-radiantly
heated individuals, even though gaping thresholds were not affected by the
source of heat. Variability, however, did not appear to be a problem in our
study due to the paired, repeated nature of the experimental design and the
fact that we assessed percentage time spent gaping to estimate the
ET50 threshold (Fig.
2). Previous studies have used the first attempt at gaping as the
gaping threshold (Chong et al.,
1973; Dupré et al.,
1986; Heatwole et al.,
1973; Parmenter and Heatwole,
1975). It is possible that assessing a simple value such as the
initial gape has much more variability inherent in the estimate, particularly
if the response is truly sigmoidal, as we have demonstrated. At the lower
temperatures, lizards spend so little time gaping that there is little effect
of increasing temperature on the time spent gaping. Furthermore, the magnitude
of gaping is much less at lower temperatures
(Fig. 4), begging the question
of how much evaporative cooling occurs when lizards are using Type I gaping.
Using an estimate such as the ET50 incorporates the entire
response over a range of temperatures and allows for an objective estimate of
the animal's overall gaping response to increasing temperature.

There was, however, a significant negative correlation between N
and body size at 6% O2, suggesting that the smaller lizards
exhibited a more typically on–off approach (i.e. a steep sigmoidal
relationship between gaping time and ambient temperature) to gaping in severe
hypoxia rather than a graded transition into increasingly more time spent
gaping. This might reflect the fact that, as the smaller-sized lizards were
undergoing more rapid changes in Tb, they responded with a
more dramatic increase in time spent gaping as their Tb
was raised above Tset and evaporative cooling mechanisms
were required.

Effect of sex on gaping threshold

Few attempts have been made to examine the role of sex on normal
thermoregulatory behaviours in reptiles outside of the breeding season (see
Lailvaux et al., 2003). Even
with pregnancy, there are inconsistent results regarding whether males and
females exhibit consistently different preferred Tb. In
one previous study, females had higher mean 24 h Tb than
did males (Sievert and Hutchison,
1989), although the reverse or lack of difference occurs just as
often, with field data often contradicting laboratory thermal gradient
experiments (Lailvaux et al.,
2003). Interestingly, we have shown that sex had a significant
effect on the gaping threshold (Table
1).Females consistently initiated gaping at a lower temperature
than males, even during hypoxia. Previously, Heatwole et al.
(1973) had shown that female
Jacky dragons had a tendency toward lower (although not significant) gaping
thresholds, a result consistent with the present study.

To the best of our knowledge, this is the first example of a sex difference
related to a physiological response to hypoxia in a reptile. If a lowered
set-point in hypoxia is truly adaptive, then it could be argued that all
lizards, regardless of sex, should lower Tb and gaping
threshold to an equal extent or as far as possible. The fact that the
difference between males and females is retained in hypoxia suggests that sex
differences in Tb regulation are of overriding importance.
A corollary of the above is that females will presumably have greater
tolerance to hypoxia if their lower gaping thresholds also translate into a
lower overall Tb regulation. Wood and Stabenau
(1998) showed that female rats
exhibit a lower Tb in hypoxia, which helped translate into
a greater tolerance to hypoxia. Survival times in hypoxic female rats were
also significantly longer than in male rats. Whether the same sensitivities
occur in reptiles is unknown.

Concluding remarks

It is apparent from the present study that bearded dragons make use of a
subtle behavioural response to effect changes in Tb. As an
ectotherm, they may not be able to artificially augment (for exceptions, see
Tattersall et al., 2004) or
decrease their overall Tb that far from ambient
temperature; however, they can use behavioural responses to serve as brakes on
thermal changes. The fact that gaping is proportionately controlled in both
duration and magnitude with respect to both ambient temperature and oxygen
strongly supports the importance of precise thermoregulatory control in as
much as other physiological and behavioural constraints will allow. It remains
to be shown exactly how effective gaping is at controlling
Tb for prolonged periods, how often this behaviour occurs
in the wild and whether it is routinely used in response to other stressors in
addition to hypoxia. Further information on the altered gaping or panting
thresholds in those species of lizards that do not routinely use panting as a
`low-cost' component to thermoregulation could be a fruitful avenue of future
research.

ACKNOWLEDGEMENTS

We would like to thank Bill Mears for providing us with the bearded
dragons, and acknowledge the excellent animal care provided by Dayle Belme,
Nicole Handley, and Sara Stewart from Brock University. This project was
funded by an NSERC operating grant to Glenn Tattersall and by an NSERC
undergraduate research assistantship (USRA) to Rebecca Gerlach.

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